PROJECT SUMMARY/ABSTRACT (RESEARCH PROJECT III) The establishment of pregnancy requires acquisition of key competencies within the embryo and female reproductive tract. Central to embryo competence is development of the trophoblast lineage, which represents the initial differentiation event during embryogenesis. Trophoblast cells are situated at the embryo-uterine interface and contribute fundamentally to the growth and survival of the embryo in the female reproductive tract. These vital tasks are accomplished through appropriate expansion of trophoblast stem (TS) cells and their accrual of specializations facilitating trophoblast modification of the uterine environment. Disruptions in trophoblast lineage development are associated with implantation failure, recurrent pregnancy loss, and a range of diseases affecting placentation, fetal growth, and postnatal development. Thus it is essential to understand molecular mechanisms underlying the regulation of TS cell renewal and differentiation. Transcriptional and epigenetic regulators control these fundamental processes, which are conserved across species utilizing hemochorial placentation. We have demonstrated the involvement of a defined set of histone modifications in the process of TS cell renewal and differentiation. We propose that Suv39h2, a histone H3K9 methyl transferase, and Kdm3a, a histone H3K9 demethylase, can act as a developmental switches regulating the TS cell stem state and acquisition of specialized trophoblast functions. Through chromatin modifications such as those engineered by Suv39h2 and Kdm3a networks of genes can be activated or silenced. In this project, three aims are proposed: 1) to evaluate the impact of histone H3K9 methylation machinery on TS cell renewal and differentiation; 2) to identify gene networks linked to dynamic histone H3K9 methylation changes during TS cell renewal and differentiation; 3) to elucidate transcription factor and co-regulator circuitry that impacts histone H3K9 methylation machinery. The investigation utilizes in vitro stem cell models and in vivo rat models to explore the role of histone H3K9 methylation in regulating the TS cell state. The proposed research provides an innovative approach to study TS cell regulation and to expand our understanding of early pregnancy loss.